In the last decades the steady increased expectation of life in developed countries has been observed. In 2000 in the USA there were 13% of people older than 65 years, in 2030 the number is being expected to increase up to 20%. The demographical change demands accelerating efforts to design an effective medical treatment for senior people. This demand refers in full measure to the cosmetic dermatology which provides with roborant, anti-aging and sun-protecting agents as well as anti-wrinkle creams. Cosmetic and pharmaceutical companies often use peptides as active ingredients in such creams. Peptides and the cosmetics on their basement have different activity: stimulation of fibroblast activity, inhibiting of the enzymes involved in collagen degradation, angiogenesis stimulation, immunomodulating, regulating melanin production, neuromuscular transmission blockage. One of the most important drawbacks for topical application of the peptides in creams is their decreased ability to penetrate into the skin. In fact, the ability to penetrate into the skin depends on various factors such as physical and chemical conditions of the compound (dissociation constant [pKa], molecular size, stability, solubility and lipophilicity coefficient); penetrating time; sustainability, thickness and skin composition, skin metabolism; area, square and duration of the application (Ranade, V. V. Drug delivery systems. 6. Transdermal drug delivery. J. Clin. Pharmacol. 1991, 31, 401-418). A peptide is considered to be a suitable tool for topical application if it corresponds to the parameters listed below, however, it must be noted that all the parameters are empirical and not universal (Guy, R. H. Current status and future prospects of transdermal drug delivery. Pharm. Res. 1996, 13, 1765-1769):                1. Molecular mass less than 500 Da        2. Lipophilic coefficient value (logarithm of the distribution coefficient for the octanol/water system) from 1 to 3.        3. Melting temperature under 200 C.        4. Good solubility in water (1 mg/ml)        5. No or few polar centers.        
Peptides used in cosmetology can be divided into four major groups: signal peptides, enzyme inhibitors, carrier peptides and blockers of neuromuscular transmission (Gorouhi F, Maibach H I. Role of topical peptides in preventing or treating aged skin. Int J Cosmet Sci. 2009 October; 31(5):327-45). Despite the similar visible physiological effect after using the peptides from the four groups, the mechanism of their action differs significantly. The peptides from the group of blockers of neuromuscular transmission are therewith considered as a safe alternative for botulotoxin injections used for treatment of age-related and mimic wrinkles. Highly specific botox has many drawbacks such as high toxicity and therefore, the necessity of the exact dose amount calculation, essential dependence of product quality on manufacturing conditions, injected usage only.
Muscle contraction is a physiological process under which muscles undergo tension—shorten or lengthen providing mechanical work. This process provides animals and humans with the ability to spontaneous and nonspontaneous movements and directly connected with digestive, respiratory, defensive, secretory and other physiological processes. Unstriated muscle is responsible for nonspontaneous movement, for example ventricle or intestinal peristalsis, changes in tonus of blood vessels and urinary bladder. Striated muscle provides spontaneous movement—spatial motion, face mimics, breath, swallowing etc. Heart work is provided by the heart muscular contraction.
Muscles are formed by multinuclear muscular fibers each of them separately is not only cellular but also physiological unit owning to the presence of such a specific “contracting” element as myofibrils. Those filaments are gathered into the clusters of the first order; several first-order clusters combine and form clusters of the second order etc. leading to the muscle formation. As muscle contraction is initiated by an impulse coming from the central nervous system, there are areas on the muscle which are innervated with synaptic terminals of neuronal axons. The junction between the neuron and muscle fiber is called neuromuscular synapse (NMS).
The mechanism of muscle contraction can be divided into several main stages. The first stage is sending a stimulus from neurons in a form of action potential. Action potential spreads along the nerve fibril to its terminals on the muscle fibers which leads to acetylcholine (Ach) neurotransmitter release from the presynaptic part of NMS into the synaptic gap. This neurotransmitter affects only a part of the muscle fiber membrane (postsynaptic part of NMS) opening various acetylcholine gated channels—acetylcholine receptors (AChRs). In response to the opening of the channels, there is an increase in sodium concentration inside the muscle fiber that leads to generation of action potential on cell membrane which is conducted longwise the muscle fiber membrane. Action potential depolarizes muscle membrane leading to calcium ions release from sarcoplasmic reticulum. Calcium ions directly initiate the process of muscle contraction. By means of calcium pump, calcium is actively pumped back into the sarcoplasmic reticulum leading to muscle relaxation.
The mechanism of acetylcholine release is well established. When neuronal impulse in a form of action potential reaches the termini of the motor neurons it opens calcium channels located on the presynaptic membrane of NMS. Local increase in intracellular calcium ion concentration is followed by calcium interaction with the proteins which provide fusion of synaptic vesicles containing neurotransmitter Ach with the plasma membrane. The process has also been studied in details and goes with the help of complex SNARE formed by the effective four-helix junction of three proteins—vesicle surface protein synaptobrevin, syntaxin and neuronal surface membrane protein SNAP-25. The complex formation leads to rapid fusion of the vesicle and plasma membranes and induces exocytosis of Ach into the synaptic gap. Ach molecules defuse throughout the gap with width 50-100 nm and reach postsynaptic membrane which is very sensitive to the transmitter due to the presence of highly specific receptors—AchRs. Their binding with Ach initiates opening of the channel; it causes dramatic influx of sodium inside the cell and a weaker flow of potassium out of the cell with the further depolarization of muscular membrane, calcium release and muscle contraction as described above.
The mechanism of neuronal impulse transmission through the postsynaptic part of NMS resulting from the binding of meurotransmitter Ach to the AChR and was also well studied. There are two major groups of AChRs—nicotinic (nAChRs) and muscarinic (mAChRs) which differ in their ability to bind with agonists. In particular, nAChRs were named for their ability to bind a natural plant alkaloid nicotine, and mAChRs—for the ability to bind muscarine alkaloid from poisonous mushrooms. NAChRs are ionotropic receptors which are permeable for specific ions after binding with a ligand. MAChR refers to a group of metabotropic receptors; it is a one-chain protein containing 7 transmembrane fragments associated with G-pritein. In this case, signal transmission after ligand binding follows many metabolic pathways.
At the molecular level nAChRs are oligomeric proteins consisting of 5 subunits. In membranes the 5 subunits are known to be pseudo symmetrically organized around the central axis where there is an ionic channel with diameter of 2.5 nm. These data were obtained for nAChR from the electrical organ of the electric ray Torpedo; this receptor has subunit composition (α1)2-β1-γ-δ [Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J Mol Biol 2005; 346: 967-89]. Up to present, 10 various subtypes of α-subunits (α1-α10) and 4 β subunits (β1-β4) are discovered. All the subtypes of α- and β-subunits (except α1- and β1-) are revealed in neuronal type nAChRs expressed mainly in neurons of the central and/or peripheral nervous system in many cases even on the presynaptic membrane [Dani J A, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol 2007; 47: 699-729]. Postsynaptic nAChRs of both animal and human neuromuscular synapses have composition (α1)2-β1-γ-δ (identical to the one for the receptor from electrical organ) but at the stage of the initial (embryotic) development of an organism. In the mature form γ-subunit is substituted with ε-[Yomoto N., Wakatsuki S., Sehara-Fujisawa A. 2005. The acetylcholine receptor gamma-to-epsilon switch occurs in individual endplates. Biochem. Biophys. Res. Commun 331:1522-1527]. In the mature muscle, only nAChR with the structure (α1)2-β1-ε-δ is responsible for binding the neurotransmitter which leads to muscle contraction. Taking into account the highly homologous structure of all the nAChR sunbunits, their spatial pentameric channel composition in the membrane is considered to be similar too. Nowadays the nAChRs region for binding with the classical agonists and competitive antagonists is also discovered: two ligand binding regions are located in the area of the contact of major N-terminal extracellular domains of two α1-and neighboring γ(ε)- and δ-subunits of the receptor approximately in the middle part towards the membrane surface.
To provide the neuron and the muscle fiber with the fast information transmission, high concentration of AChR in essential areas of postsynaptic membrane of neuromuscular junction is needed. Therefore, aggregation of AChRs is crucial for normal functioning of neuromuscular junction [Hoch W. 1999. Formation of the neuromuscular junction. Agrin and its unusual receptors. Eur. J. Biochem. 265:1-10]. There are several proteins involved in the process of aggregation, mainly agrin, rapsin and kinase MuSK (Muscle-Specific Kinase). Developed earlier and being created nowadays new agents blocking neuromuscular transmission are aimed at disruption of normal functioning of either presynaptic membrane of NMS or postsynaptic part of a synapse.
Cosmetic industry has undertaken several various efforts to develop new compounds for the topic application during the treatment of mimic wrinkles to avoid side effects observed after botulinum toxin injections (Lupo M P, Cole A L. Cosmeceutical peptides. Dermatol Ther. 2007, September-October;20(5):343-9). Up to present, several peptide compounds are known to block neuromuscular transmission (Table 1)
TABLE 1Amino acid sequence of   the peptides blocking neuromuscular  transmission, and their biological activities.Amino acidNamesequenceActivityArgirelineAc-Glu-Glu-Inhibits formation Met-Gln-Arg-of the complex Arg-NH2SNARE and(SEQ ID neurotransmitterNO: 49)release LeuphasylH-Tyr-D-Ala-Mimics the action Gly-Phe-Leu-of enkefalin - OHdecreases neuronalexcitation VialoxH-Gly-Pro-Arg-Competitive Pro-Ala-NH2 antagonist of nAChR(SEQ IDNO: 50) Syn-AkeH-β-Ala-Pro-Competitive Dab-NHBzlantagonist of nAChR Inylinenot publishedCompetitive  antagonist of MuSK
Peptide argireline is the top one within “cosmetic analogues of botox” (Blanes-Mira C., et al. A synthetic hexapeptide (Argireline) with anti-wrinkle activity. Int J Cosmet Sci. 2002; 24(5): 303-310). Appearing at the beginning of 2000, it rapidly gained the popularity—today it is used in many cosmetic drugs aimed at smoothing mimic wrinkles. Six amino acids of argireline repeat the protein fragment SNAP 25 necessary for synaptic axonal vesicle binding with the presynaptic membrane. In an axon, argireline competes with protein SNAP25 and inserts into the temporary complex SNARE instead of it—this complex is formed from several membrane proteins directly before synapse binds with the membrane, and it is necessary for successful exocytosis. A deficient complex cannot provide a good junction of vesicle with membrane, as a result transmitter release does not occur and muscle does not receive a signal to contract and stay relaxed.
Argireline analogues obtained from protein SNAP 25 and aimed at inhibiting neuromuscular transmission at synaptic level by competing with SNAP 25 for complex SNARE formation, are described in patents EP 1180524  WO9734620.
Topic 30-day use of cream containing 10% of argireline studied on volunteers leads to 30% decrease in the depth of mimic wrinkles in comparison with 10% exposure of Placebo. Pentapetide Leuphasyl developed by Lipotech company (Spain) mimics enkephalin action decreasing neuronal excitation by inhibiting Ca2+-influx throughout the membrane and decreasing Ca2+-dependant transmitter release. Use of Leuphasyl in a combination with argireline enhances argireline effect by 1.5-fold.
Inyline is an acetyl hexapeptide developed by Lipotech company (Spain) (WO2011/009626) was created basing on the predicted structure of the binding area of agrin to MuSK (Muscle-Specific Kinase) obtained by molecular modeling. Inyline works as a competitive antagonist of MuSK at binding with agrin, which is essential for muscle contraction. Postsynaptic mechanism of decreasing muscle contraction helps to avoid appearance of mimic wrinkles. Studies on volunteers have shown that 28-day administration of 5% Inyline solution leads to reduction in the depth of the wrinkles in the area of “crows feet” on 14.9%.
Vialox (Pentapharm company, Switzerland) is a pentapeptide fragment of the neurotoxin waglerin-1 from the venom of the Temple Viper. Vialox has a curare-like activity and it is a competitive antagonist of the nAChR. When the peptide binds the receptor sodium ion channel keeps closed which disrupts neuronal impulse transmission and does not allow muscles to contract. Patent EP1809652 describes peptide antagonists of the acetylcholine receptor which work postsynaptically following the vaglerine-1-like mechanism by blocking neuronal transmission and preventing wrinkle appearance. Vialox can be used together with other cosmetic peptides (WO 2006/069608).
28-day application of the cream containing 5% Vialox reduces wrinkle size up to 49% and skin roughness up to 47%.
One more active cosmetic peptide Syn-ake (Pentapharm, Switzerland) is a reversible antagonist of the muscle-type nicotinic acetylcholine receptor (mnAChR). This tripeptide acts in a manner similar to waglerin-1, which prevents acetylcholine binding with the receptor and the channel activation. Therefore, the muscles stay relaxed (WO 2006/047900). The studies performed on volunteers showed that the 28-day application of the cream containing 4% Syn-Ake decreases depth of wrinkles on foreheads up to 52%.
Thus, the competitive antagonists of the mnAChR Vialox and Syn-Ake are the most effective peptides with anti-wrinkle activity.
However, none of the compounds developed by cosmetic or pharmaceutical companies can inhibit muscle contraction with the effectiveness similar to botulinum toxin. Therefore, the necessity for creating new compounds capable to inhibit muscular contraction and reaching better results in decreasing and emolliating wrinkles especially mimic ones, still exists.